The DNA sequence and biological annotation of human chromosome 1.

The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.

Biosynthesis of pro-C3, a precursor of the third component of complement.

A precusor of the third component of complement, pro-C3, was detected in studies of cell-free synthesis and intracellularly in homogenates of liver tissue cultures. The molecular weight of pro-C3 was indistinguishable from that of intact native C3 secreted in vitro by liver or peritoneal macrophages, but its structure was different. Pro-C3 is a single polypeptide chain, whereas C3 secreted by cells in culture consists of two polypeptide chains (mol wt 120,000 and 76,000) linked by disulfide bonds.

Accessory cell function of human B cells. I. Production of both interleukin 1-like activity and an interleukin 1 inhibitory factor by an EBV-transformed human B cell line.

In the present paper we report that the ROHA -9 cell line, an Epstein-Barr virus (EBV)-transformed human B cell line with accessory cell capabilities, constitutively secretes a soluble factor with the biochemical and biological characteristics of human monocyte-derived IL-1. The IL-1 derived from ROHA -9 augmented murine thymocyte proliferation and enhanced the proliferative response of human T lymphocytes to concanavalin A (Con A). The ROHA -9-derived IL-1 activity eluted from Sephacryl S-200 in two peaks, at 15- 18K and 32- 35K mol wt, eluted from DEAE-Sephacel at 50-80 and 110-130 mM NaCl, and showed charge heterogeneity with peaks at pI 7.3, 6.1, and 4.1 on isoelectrofocusing (IEF). These findings suggest that B cells may elaborate an IL-1-like activity. During the logarithmic growth of ROHA -9 cells, a inhibitory factor that inhibited the response of mouse thymocytes to IL-1 was also produced. This factor had a mol wt of 95K on Sephacryl S-200, eluted at 150 mM NaCl on DEAE-Sephacel and showed a peak of pI 4.7 on preparative IEF. The inhibitory factor appeared to be selective in its effects on IL-1 responses, since it did not inhibit the activity of IL-2 on mouse thymocytes or on the growth of the IL-2-dependent CT6 cell line. This "contra-IL-1" inhibited the response of murine thymocytes to suboptimal (1 microgram/ml) but not optimal (10 micrograms/ml) doses of Con A and the response of human peripheral blood lymphocytes to streptolysin O ( SLO ) or to alloantigens. Moreover, the factor could be absorbed by mouse thymocytes but not by CT6 cells, and such thymocytes pretreated with contra-IL-1 failed to response to IL-1. Although this inhibitor is the product of a transformed B cell line, it may be representative of regulatory substances that normally control IL-1 activities either at the extracellular or intracellular level.

Cooperative interaction of factor B and other complement components with mononuclear cells in the antibody-independent lysis of xenogeneic erythrocytes.

Synergistic cytotoxicity is a term used to describe a cytotoxic system in which xenogeneic erythrocyte target cells are lysed in the presence of nonimmune human mononuclear effector cells and antibody-depleted normal human serum. Neither the mononuclear cells nor the serum alone are cytolytic to the target erythrocytes. Previous studies have shown that the serum activity is not immunoglobulin and is heat-labile, suggesting a similarity to serum complement. In this report, sera deficient in various complement components as well as highly purified single complement components were tested with whole mononuclear cell populations and purified monocytes and lymphocytes to further characterize this cytotoxicity system. Whole mononuclear cell populations failed to mediate target cell lysis in sera deficient in C5 or factor B. However, C3-deficient serum, even in the presence of anti-C3 antibody, supported synergistic cytotoxicity normally. Purified lymphocytes were also normally cytotoxic in C3-deficient serum but failed to lyse targets in sera deficient in C5, C7, C8, or depleted of factor B. Purified monocytes failed to lyse the target cells only in factor B-depleted serum and could lyse the target cells in serum-free medium when purified factor B alone was added. Monocyte-mediated cytotoxicity induced by factor B was inhibited 73-100% by adding lymphocytes back to the purified monocytes. Thus, both lymphocytes and monocytes can serve as effector cells in this form of cytotoxicity but require cooperative interaction with different sets of complement components. In addition, lymphocytes can modulate the monocyte-mediated form of target cell lysis associated with factor B.

Genetic defect in biosynthesis of the precursor form of the fourth component of complement.

Under cell-free conditions, liver polysomes from guinea pigs genetically deficient in the fourth component of complement (C4) did not synthesize pro-C4 (the precursor of C4), but did synthesize nascent C4 polypeptides which remained polysome bound. The defect was specific for pro-C4 synthesis since the amounts of total protein and albumin synthesis and release from C4-deficient polysomes were similar to that in normal guinea pig liver polysomes.

Identifying packaging errors.

Common medical device packaging problems are identified here together with their cause and the remedy. The discussion examines the causes of poor heat seal, misleading results from the measurement of seal strength, blister flange curl, damage to sterile barrier system and failure of the ethylene oxide sterilisation process.

Mitochondrial myopathy with succinate dehydrogenase and aconitase deficiency. Abnormalities of several iron-sulfur proteins.

Recently, we described a patient with severe exercise intolerance and episodic myoglobinuria, associated with marked impairment of succinate oxidation and deficient activity of succinate dehydrogenase and aconitase in muscle mitochondria (1). We now report additional enzymatic and immunological characterization of mitochondria. In addition to severe deficiency of complex II, manifested by reduction of succinate dehydrogenase and succinate:coenzyme Q oxidoreductase activities to 12 and 22% of normal, respectively, complex III activity was reduced to 37% and rhodanese to 48% of normal. Furthermore, although complex I activity was not measured, immunoblot analysis of complex I showed deficiency of the 39-, 24-, 13-, and 9-kD peptides with lesser reductions of the 51- and 18-kD peptides. Immunoblots of complex III showed markedly reduced levels of the mature Rieske protein in mitochondria and elevated levels of its precursor in the cytosol, suggesting deficient uptake into mitochondria. Immunoreactive aconitase was also low. These data, together with the previous documentation of low amounts of the 30-kD iron-sulfur protein and the 13.5-kD subunit of complex II, compared to near normal levels of the 70-kD protein suggest a more generalized abnormality of the synthesis, import, processing, or assembly of a group of proteins containing iron-sulfur clusters.

Purification and further characterization of a non-tumor necrosis factor alpha or beta differentiation-inducing cytokine, P48.

In this report, we present the further characterization and purification of a cytokine differentiation factor, termed P48, which unlike previously described differentiation factors is antigenically unrelated to tumor necrosis factor alpha (TNF-alpha), tumor necrosis factor beta (TNF-beta), and gamma interferon. HL-60 cells and phorbol diester-resistant HL-60-1E3 cells exposed to conditioned medium from Reh cells mature along the monocyte/macrophage pathway, as assessed by several assays (express nonspecific esterase, produce superoxide anion, morphologically resemble monocytes, mediate phorbol diester-triggered extracellular cytolytic activity). Reh cell conditioned medium is antiproliferative toward a panel of cell lines, is not nonspecifically cytotoxic, has no antiviral or colony-stimulating factor activities, and is not affected by exposure to insolubilized anti-gamma interferon. A 48-kDa glycoprotein (P48) which mediates this differentiation factor activity has been purified to homogeneity from Reh cell conditioned medium, and a polyclonal neutralizing antiserum has been produced. P48 activity is not blocked by either anti-TNF-alpha and anti-TNF-beta and on Western blot analysis is antigenically distinct from TNF-alpha and TNF-beta. In addition, polyclonal anti-P48 does not block either TNF-alpha or TNF-beta activities or recognize either on Western blots. Unlike gamma interferon, colony-stimulating factor, TNF-alpha, or TNF-beta, P48 reverses phorbol diester resistance of HL-60-1E3 cells. These studies present strong evidence for the existence of a previously unrecognized cytokine which, unlike other reported differentiation factors, is antigenically unrelated to TNF-alpha or TNF-beta. P48 may play an important role in growth and development of normal and abnormal (leukemic) hematopoietic and nonhematopoietic cells.

Enhancement of phorbol diester-induced HL-60-mediated cytotoxicity by retinoic acid, dimethyl sulfoxide, and 5-azacytidine.

Both peripheral blood monocytes and neutrophils are known to be capable of lysing a variety of extracellular tumor and non-tumor cell targets. The HL-60 human promyelocytic leukemia cell line has served as a useful model of human granulocyte and macrophage differentiation in studies from many laboratories. We have previously reported that phorbol diesters, which induce differentiation along the macrophage pathway, stimulate HL-60 cells to become strikingly cytotoxic to a variety of red cell targets. We now report that agents known to differentiate HL-60 along the granulocyte pathway (retinoic acid, dimethyl sulfoxide, 5-azacytidine) do not, in themselves, induce HL-60 to become cytotoxic. However, previous exposure (3-5 days) to these granulocyte pathway active agents markedly enhances phorbol diester-triggered killing. This enhancement is particularly striking at decreased effector:target ratios (as low as one effector per five targets) and is also demonstrated by a shift to lower concentrations of the phorbol diester dose-response curve. Retinoic acid is the most effective of the three agents tested, although priming (previous exposure) with dimethyl sulfoxide or 5-azacytidine also markedly enhances killing. These studies demonstrate that HL-60-mediated killing may be dissected pharmacologically into at least two distinct steps and further support the utility of this model system in studies of the development of macrophage-like cytotoxic cells. This system has also proven to be useful in the characterization of cytokines which mimic the differentiation effects of retinoic acid and dimethyl sulfoxide (J. A. Leftwich and R. E. Hall, manuscript in preparation).

Cell proliferation kinetics of MCF-7 human mammary carcinoma cells in culture and effects of tamoxifen on exponentially growing and plateau-phase cells.

MCF-7 human mammary carcinoma cells were inoculated into 150-sq cm flasks at 3 X 10(5) cells/flask, and after a lag period of about 48 hr, these cells grew exponentially for 5 days with a mean population doubling time of about 24 hr. During exponential growth, 80 to 90% of cells were in the "rapidly cycling" pool, the clonogenic fraction was 50 to 60%, and the mean percentage of cells in the G0-G1, S, and G2 + M phases of the cell cycle was 48.9 +/- 0.6% (S.E.), 39.4 +/- 0.6%, and 11.6 +/- 0.3%, respectively. These parameters changed rapidly between Days 7 and 13 when plateau phase was reached. Between Days 13 and 18, 74.8 +/- 0.7% of cells were in G0-G1, 15.3 +/- 0.4% were in S, and 9.8 +/- 0.6% were in G2 + M phase. Only about 30% of these cells were cycling rapidly, and the clonogenic fraction had fallen to less than 10%. Tamoxifen induced a dose-dependent decrease in the growth rate of exponentially growing cells, which was accompanied by a dose-dependent increase in percentage of G0-G1-phase cells, and a decline in percentage of S-phase cells. At doses greater than or equal to 10 microM, a 24-hr pulse of tamoxifen was cytotoxic to exponentially growing cells. Plateau-phase cells were less sensitive to these effects of tamoxifen. In an attempt to define the kinetic basis of the G0-G1 accumulation induced by tamoxifen, asynchronous MCF-7 cells were pretreated for 42 hr with various doses of tamoxifen, and the rate of efflux of cells from the G0-G1 phase of the cell cycle was assessed by blocking their reentry into G1 with ICRF 159. Following treatment of control cultures with ICRF 159, two populations of cells were distinguished by their rates of efflux from G0-G1 phase. The majority of cells left G0-G1 rapidly with a mean t1/2 of 2.3 hr ("rapidly cycling" cells). However, about 18% of cells had a much slower rate of exit with a mean t1/2 of about 30 hr ("slowly cycling" cells). Pretreatment with tamoxifen resulted in a dose-dependent decrease in the proportion of rapidly cycling cells and an increase in the proportion of cells with slow G1 transit times. Although this appeared to be the predominant effect, tamoxifen also decreased the rate at which the slowly cycling cells traversed G1. Simultaneous treatment with estradiol returned these parameters to control values at doses of tamoxifen less than or equal to 5 microM, partially reversed the effect of 7.5 microM tamoxifen, but was without effect on the arrest of cell cycle progression induced by 10 microM tamoxifen. It is concluded that cells accumulate in G0-G1 following tamoxifen treatment due to an increase in the proportion of slowly cycling cells at the expense of a population of rapidly cycling cells, which appear to be relatively uninfluenced by the drug.

Resistance to natural killer cell-mediated cytolysis by a pleiotropic drug-resistant human erythroleukemia (K562-R) cell line.

K562-R, a pleiotropic drug-resistant cell line established in vitro, and K562-S, the chemotherapy-sensitive parent line, were used as targets in the natural killer cell (NK) system. At each of the effector:target ratios studied, the K562-R demonstrated a decrease in their susceptibility to both unstimulated and interferon-activated NK cells, as compared to the K562-S line. This difference did not appear to be related to a variable expression of NK target structures, as the number of effector:target conjugates was similar, and both lines competed equally well in cold target inhibition experiments. The K562-R cells were not resistant to complement or monocyte-mediated killing, suggesting a relatively specific resistance to cell-mediated killing. These results are compatible with an NK postbinding defect in the K562-R cells and suggest that greater tumorigenicity for pleiotropic drug-resistant cells may in part be due to altered susceptibility to host defense.

Characterization of a K562 multidrug-resistant cell line.

A daunorubicin-resistant variant of the K562 human leukemia cell line (K562-R), which demonstrates cross-resistance to other anthracycline antibiotics and Vinca alkaloids, has been developed in vitro by continuous exposure to daunorubicin. Cross-resistance to anthracyclines and Vinca alkaloids is reversed when cells are exposed to drugs in the presence of verapamil, a calcium channel blocker. The K562-R cell line overexpresses a 4.5-kilobase mRNA, which is thought to code for the Mr 170,000 membrane glycoprotein associated with multidrug resistance. Transport studies indicate reduced intracellular accumulation and retention of daunorubicin in the K562-R cells as compared to the parent cell line. These studies further suggest the presence of distinct cellular pools composed of both rapidly and slowly exchanging drug, with the rapidly exchanging pool being more pronounced in the resistant line. The development of multidrug resistance in the K562-R cell line is also associated with the overexpression of five different cell surface membrane proteins ranging in molecular weight between 50,000 and 210,000, whose function remains to be defined.

Differential sensitivity of human breast cancer cell lines to the growth-inhibitory effects of tamoxifen.

In eight estrogen receptor (ER)-positive breast cancer cell lines (including three sublines of MCF-7) and five ER-negative breast lines, the action of the nonsteroidal antiestrogen, tamoxifen, was studied, and the concentrations of ER and antiestrogen binding site were measured. The concentration of antiestrogen binding site was significantly [P less than 0.005] greater in ER-positive cells [236,600 +/- 29,900 (SE) sites/cell] than in ER-negative cell lines [66,600 +/- 16,800 sites/cell]. In ER-positive cell lines, a cell cycle phase-specific growth-inhibitory effect, 20% inhibitory dose less than 0.1 to 1.0 microM, was seen which was shown for some representative cell lines to be estrogen reversible. Within this group of cell lines, the degree of tamoxifen-induced inhibition of growth correlated with control population doubling time, but not ER or antiestrogen binding site concentration. The changes in cell cycle kinetic parameters characteristic of all ER-positive lines were a decrease in percentage of S-phase cells and a corresponding increase in percentage of G0-G1 cells. In all cell lines, 5 to 12.5 microM tamoxifen caused cytotoxicity, and this was shown to be estrogen-irreversible in 3 representative cell lines; moreover, estradiol synergistically enhanced the cytotoxic effects of tamoxifen under some experimental conditions. The cell cycle effects of tamoxifen in three ER-negative cell lines (Hs0578T, MDA-MB-231, MDA-MB-330) were decreased proportions of G0-G1 cells with an increase in percentages of S and G2+M cells. These results implied that the mechanism of tamoxifen cytotoxicity may differ in ER-positive and ER-negative breast cancer cells. However, although the ER-negative BT-20 line was much less sensitive to tamoxifen than were the ER-positive cells, growth inhibition and cytotoxicity in this line were associated with a slight decrease in percentage of S-phase cells. These results confirm that ER-positive breast cancer cells are more sensitive (4- to greater than 75-fold) than ER-negative breast cells to the growth-inhibitory effects of tamoxifen and demonstrate that, in all ER-positive cells, growth inhibition and cytotoxicity are accompanied by characteristic changes in cell cycle kinetic parameters. In contrast, different mechanisms may be involved in the effects of tamoxifen on different ER-negative cell lines.

HL-60-1E3, a novel phorbol diester-resistant HL-60 cell line.

The HL-60 (human promyelocytic leukemia) cell line has proved to be a useful model for the study of the expression of cellular functions and markers associated with hematopoietic differentiation. We report here the development and initial characterization of a novel, differentiation-resistant HL-60 cell line (HL-60-1E3) which was established by cloning the parent HL-60 line in the absence of mutagens or differentiation-inducing agents. HL-60-1E3 exhibits markedly reduced phorbol diester-induced expression of extracellular cytolytic activity, nonspecific esterase, phagocytosis, and surface Mo1 antigen. In addition, dimethyl sulfoxide-induced expression of both Mo1 and Mo2 is markedly reduced. However, if HL-60-1E3 is exposed to dimethyl sulfoxide, it acquires appreciable phorbol diester-triggered cytolytic activity and production of superoxide anion (O2-). Phorbol diester receptor number and dissociation constant (Kd) obtained by Scatchard analysis are not significantly different for the two cell lines. The HL-60-1E3 cell line should provide a useful adjunct to other cell lines used in the study of normal myeloid and leukemic cell differentiation, as well as the study of cytokine maturation factors and oncogene expression.

Expression and regulation of retinoic acid receptors in human breast cancer cells.

Retinoic acid is known to inhibit mammary carcinogenesis in rodents and to inhibit proliferation and steroid hormone receptor gene expression in human breast cancer cells. Since these effects are likely to be mediated by nuclear retinoic acid receptors (RARs) the present study was initiated to determine the expression and regulation of RARs in human breast cancer cell lines. Differential cellular gene expression of the RARs was determined by Northern blot analysis of total RNA prepared from 5 ER+ and 6 ER- cell lines. RAR alpha was detected as mRNA species of 2.7 and 3.4 kilobases in all cell lines and the level of gene expression was greater in ER+ cell lines (P less than 0.001). RAR beta mRNA (3.7 kilobases) was detected in seven of the eleven lines tested and was expressed most commonly in ER- cell lines. RAR gamma mRNA was expressed in all cell lines as a transcript of 3.4 kilobases at levels that were similar in both ER+ and ER- cell lines. Retinoic acid failed to regulate the expression of the RAR alpha and RAR gamma genes. The effect of steroid hormones on RAR alpha and RAR gamma mRNA levels was also examined. In four PR+ cell lines (T-47D, BT 474, MCF-7M, and MDA-MB-361), progestins markedly decreased RAR alpha mRNA levels. The progestin effect on RAR alpha levels in T-47D cells was detectable at concentrations of 0.05 nM and was maximal at 1 nM 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione ORG 2058, whereas dihydrotestosterone and dexamethasone were without effect. RAR alpha and RAR gamma mRNA levels were rapidly decreased by progestin, and the effect was maximal 3-6 h after ORG 2058 treatment. However, the mRNA loss was transient, and recovery of RAR alpha and RAR gamma mRNA levels was noted 12-24 h after retinoic acid treatment. Although RAR gamma mRNA returned to control levels by 24 h, the decrease in RAR alpha mRNA was maintained at around 50% control until at least 48 h. In summary, RAR alpha and RAR gamma were expressed in all human breast cancer cell lines and were regulated by progestins in the PR+ T-47D cell line. The previously reported ability of retinoic acid to down-regulate PR mRNA and the present demonstration that progestins down-regulate RAR alpha and RAR gamma mRNA suggest that mutual regulation may be a mechanism through which PR and the RARs interact in human breast cancer cells.

Effect of medroxyprogesterone acetate on proliferation and cell cycle kinetics of human mammary carcinoma cells.

The effect of medroxyprogesterone acetate (MPA) on breast cancer cell proliferation kinetics was investigated in ten human breast cell lines growing as monolayer cultures. Significant inhibition of growth occurred only in the estrogen receptor-positive, progesterone receptor-positive cell lines, T-47D, MCF-7, ZR 75-1, BT 474, and MDA-MB-361. Among these cell lines sensitivity to MPA varied widely; concentrations required for 20% inhibition of growth ranged from 0.04 nM for T-47D to greater than 100 nM for ZR 75-1 cells. Furthermore, although the most sensitive line, T-47D, had the highest level of PR, sensitivity to MPA was not correlated with PR levels among the responsive cell lines. More detailed studies were undertaken with the T-47D cell line. The growth-inhibitory response was confined to the progestins: MPA, ORG 2058, R5020, and progesterone, while androgens, estrogens, and glucocorticoids were without effect over the same concentration range (0.1-100 nM). MPA-induced growth inhibition was associated with a significant decrease in the proportion of S-phase cells with an accumulation of cells in the G0-G1 phase of the cell cycle. Cells began to accumulate in G0-G1 after 12 h of drug treatment and the effect was maximal by 24 h, i.e., maximal effects were observed during the first cell cycle following drug treatment. By contrast, significant accumulation in G0-G1 required exposure of MCF-7 cells to MPA for at least two cell cycle times, i.e., 48 h and the effect was still increasing at 96 h. Stathmokinetic studies revealed that in both cell lines accumulation in the G0-G1 phase was due to an MPA-induced increase in the G1 transit time. These data indicate that MPA and other progestins have direct growth inhibitory effects on estrogen receptor-positive and progesterone receptor-positive human breast cancer cells in vitro and these effects can be accounted for by a decrease in the rate at which cells traverse the G1 phase of the cell cycle.

Altered regulation of c-myc in an HL-60 differentiation resistant subclone, HL-60-1E3.

HL-60 cells treated with phorbol dibutyrate (PDBu) differentiate into cells which functionally and morphologically resemble macrophages (G. Rovera, D. Santoli, and D. Damskey, Proc. Natl. Acad. Sci. USA, 75: 2779-2783, 1979; E. Huberman and M.F. Callahan, Proc. Natl. Acad. Sci. USA, 76:1293-1298, 1979). This differentiation involves modulation of the expression of several cellular oncogenes. However, the significance of the temporal relationships between differentiation events and specific oncogene expression are not known. Others have reported that transcriptional down regulation of c-myc occurs early in the differentiation of HL-60 cells (R.D. Dalla-Favera et al., Haematol. Blood Transfusion, 28: 247-253, 1983; L.E. Grosso and H.C. Pitot, Biochem. Biophys. Res. Commun., 119: 473-480, 1984). To determine the significance of the regulation of c-myc during HL-60 maturation, we performed parallel PDBu induction studies analyzing the kinetics of expression of c-myc, cell cycle frequency distribution, cytotoxic effector activity, and clonogenic potential in HL-60 cells and in a partial-differentiation resistant HL-60 clone (HL-60-1E3) (J. A. Leftwich, P. Carlson, B. Adelman, and R. E. Hall, Cancer Res., 47: 1319-1324, 1987). We report that PDBu stimulation results in early c-myc transcriptional down regulation in the HL-60-1E3 clone cells with the same kinetics as has been previously reported for HL-60 parental cells (R. D. Dalla-Favera et al., Haematol. Blood Transfusion, 28: 247-253, 1983; L. E. Grosso and H. C. Pitot, Biochem. Biophys. Res. Commun., 119: 473-480, 1984). However, reexpression of c-myc occurs 15 hours postinduction in HL-60-1E3 but not parental cells. This reexpression is maintained through 30 h of stimulation and correlates with a lack of terminal commitment as assessed by an increase in clonogenic potential and the inability of these cells to acquire cytotoxic function. Sequential stimulation of HL-60-1E3 cells with DMSO and PDBu overcomes the block in macrophage differentiation (J. A. Leftwich, P. Carlson, B. Adelman, and R. E. Hall, Cancer Res., 47; 1319-1324, 1987). Such treatment results in a transient reexpression of c-myc at 15 h after PDBu treatment, and the complete downregulation of c-myc 24 h postinduction. These data suggest that the reported early decrease in c-myc transcripts following PDBu stimulus in HL-60 cells is not sufficient to commit these cells to macrophage-like terminal differentiation. Late regulation of c-myc gene expression may be an important additional component of the regulatory mechanisms which allow HL-60 cells to complete this program.

Tissue distribution of cytochrome c oxidase isoforms in mammals. Characterization with monoclonal and polyclonal antibodies.

Monoclonal and polyclonal antibodies specific to the two isoforms of subunit VIa of bovine cytochrome c oxidase were generated and used to study the tissue distribution of this subunit pair in beef, human and rat. The so-called H-(heart) form was found exclusively in heart and skeletal muscle, whereas the so-called L-(liver) form was the only isoform present in brain, kidney, liver and smooth muscle. Little or no L-form was detected in skeletal muscle. In bovine heart no subunit VIa-L was detected, while in human heart the subunit VIa-H and VIa-L isoforms were present in roughly equal proportions. These results imply that, in humans, the deficiency of a subunit VIa isoform may have a different effect on the physiology of heart then on the physiology of skeletal muscle.

Initial characterization of a cytokine which induces differentiation and cytolytic activity in HL-60 promyelocytic leukemia cells: evidence that the cytokine is distinct from other known differentiation-active cytokines.

In this paper we report that, like dimethyl sulfoxide (DMSO), retinoic acid (RA), and conditioned medium (CM) from lectin-stimulated mononuclear leukocytes, CM from a human null cell leukemia line (Reh) induces HL-60 promyelocytic leukemia cells to respond in an enhanced manner to phorbol diester (PDE). Furthermore, Reh-CM induces PDE-resistant HL-60-1E3 cells to respond to PDE and lyse target cells. Additionally, both HL-60 and HL-60-1E3 cells exposed to Reh-CM for 3 days produce superoxide anion and express cell surface antigens present on mature mononuclear phagocytes. No colony-stimulating factor (CSF) or interferon (IFN) activity was detected in Reh-CM, and differentiation activity (DA) was not removed from Reh-CM by insolubilized anti-IFN gamma. While Reh-CM is antiproliferative against a panel of cell lines, its spectrum of activity is different than tumor necrosis factor (TNF) alpha, and neither TNF alpha nor TNF beta inhibit proliferation of HL-60-1E3 or induce these cells to respond to PDE. The differentiation factor (DF) material has been partially purified by ammonium sulfate precipitation and is non-dialyzable; unstable to heat, acid, or alkali treatment; and the activity is not blocked by anti-IL-6 or anti-IFN alpha. The data presented in this paper suggest the presence of a differentiation-inducing factor which is distinct from CSF, IFN alpha or -gamma, TNF alpha, or -beta, or IL-6, which may play a role in the differentiation of malignant (leukemic) and normal cells of the myelomonocytic lineage.

A monoclonal antibody (RH1-38) which inhibits multiple systems of cell-mediated cytotoxicity--I. Identification of a novel epitope on a killer cell surface bimolecular complex important in the cytotoxic response.

This paper presents the initial characterization of a mouse monoclonal antibody (RH1-38) which blocks, in the absence of complement, three different systems of cell-mediated cytotoxicity. This monoclonal antibody markedly inhibits cytotoxicity mediated by human natural killer cells, a monocyte-like cell [phorbol myristate acetate (PMA) stimulated HL-60], and cytotoxic T-lymphocytes generated in a mixed leukocyte reaction. RH1-38 is not nonspecifically toxic to cells since antibody-dependent cellular cytotoxicity was not inhibited and viability as assessed by trypan blue exclusion was not affected. Inhibition is specific since control hybridoma culture supernatants, parent (NS-1) ascites supernatant, monoclonal anti-HLA and normal mouse IgG were not significantly inhibitory. In the NK system, the inhibitory effect appears to be due to binding of monoclonal antibody to effector cell surface since exposure of targets to antibody followed by washing yielded no inhibition of killing. Inhibition requires the antigen-binding portion of the antibody molecule and thus appears to be related to steric hindrance of an effector cell surface molecule which is important in the expression of cell-mediated cytotoxicity. Immunoprecipitation of surface-radioiodinated membranes from PMA-stimulated HL-60 cells and analysis on sodium dodecyl sulfate-polyacrylamide gels revealed a bimolecular complex (195,000 and 125,000 daltons) without significant change under reducing conditions. Control immunoprecipitates yielded no peaks of activity. This monoclonal antibody should serve as a useful probe of the function and biochemistry of a killer cell surface antigen important in the expression of cell-mediated cytotoxicity. Since RH1-38 inhibits cytotoxicity mediated by at least three apparently unrelated effector cells, the relevant antigen may be part of a common mechanistic step. As the companion paper demonstrates, this monoclonal antibody does not affect the conjugation step, but appears to block a late step in the NK cytolytic mechanism. Thus, RH1-38 recognizes either an epitope district from previously-described anti-LFA-1 antibodies or alternatively recognizes a distinct functional killer cell surface molecule.